Antidepressants increase neural progenitor cells in the humanhippocampus

Maura Boldrini1,5,7, Mark D. Underwood1,5, René Hen1,3,4,6, Gorazd B. Rosoklija1,5,8,Andrew J. Dwork1,2,5, J. John Mann1,5, and Victoria Arango1,5

1Department of Psychiatry, Columbia University2Department of Pathology and Cell Biology, Columbia University3Department of Neuroscience, Columbia University4Department of Pharmacology, Columbia University5Division of Molecular Imaging and Neuropathology, New York State Psychiatric Institute6Division of Integrative Neuroscience, New York State Psychiatric Institute7Department of Neurological and Psychiatric Sciences, University of Florence, Italy8Macedonian Academy of Sciences & Arts

AbstractSelective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs) increaseneurogenesis in the dentate gyrus (DG) of rodents and nonhuman primates. We determinedwhether SSRIs or TCAs increase neural progenitor (NPCs) and dividing cells in the human DG inmajor depressive disorder (MDD).

Whole frozen hippocampi from untreated subjects with MDD (N = 5), antidepressant-treatedMDD (MDDT, N = 7), and controls (C, N = 7) were fixed, sectioned and immunostained forNPCs and dividing cell markers (nestin and Ki-67 respectively), NeuN and GFAP, in single anddouble labeling. NPC and dividing cell numbers in the DG were estimated by stereology. Clinicaldata were obtained by psychological autopsy and toxicological and neuropathological examinationperformed in all subjects.

NPCs decreased with age (p = 0.034). Females had more NPCs than males (p = 0.023). Correctingfor age and sex, MDDT receiving SSRIs had more NPCs than untreated MDD (p ≤ 0.001) andcontrols (p ≤ 0.001), NPCs were not different in SSRIs- and TCAs-treated MDDT (p = 0.169).Dividing cell number, unaffected by age or sex, was greater in MDDT receiving TCAs than inuntreated MDD (p ≤ 0.001), SSRI-treated MDD (p = 0.001) and controls (p ≤ 0.001). The NPCsand dividing cells increase in MDDT was localized to the rostral DG. MDDT had a larger DGvolume compared with untreated MDD or controls (p = 0.009).

Address correspondence to: Maura Boldrini, MD, PhD, Department of Psychiatry, Division of Molecular Imaging andNeuropathology, Columbia University College of Physicians & Surgeons, New York State Psychiatric Institute, 1051 Riverside Drive,Box 42, New York, NY 10032 USA, Phone: 212-543-5440, Fax: 212-543-6017, Email: mb928@columbia.edu.

Disclosure/Conflict of Interest: The authors Maura Boldrini, Mark D. Underwood, Andrew J. Dwork, Gorazd B. Rosoklija andVictoria Arango declare that, except for income received from their primary employer, no financial support or compensation has beenreceived from any individual or corporate entity over the past three years for research or professional service and there are no personalfinancial holdings that could be perceived as constituting a potential conflict of interest.Dr. Rene' Hen receives compensation as a consultant for BrainCells, Inc., PsychoGenics, Inc., and AstraZeneca in relation to thegeneration of novel antidepressants.Dr. J. John Mann received grants from GlaxoSmithKline and Novartis.

NIH Public AccessAuthor ManuscriptNeuropsychopharmacology. Author manuscript; available in PMC 2010 April 1.

Published in final edited form as:Neuropsychopharmacology. 2009 October ; 34(11): 2376–2389. doi:10.1038/npp.2009.75.

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Antidepressants increase neural progenitor cell number in the anterior human dentate gyrus.Whether this finding is critical or necessary for the antidepressants effect remains to bedetermined.

KeywordsAdult neurogenesis; Ki-67; nestin; major depressive disorder; SSRIs; tricyclic antidepressants

IntroductionNeurogenesis has been detected in the adult hippocampus of rodents (Miller andNowakowski 1988; Kee et al, 2002; Santarelli et al, 2003), nonhuman primates (Kornackand Rakic 1999; Gould et al, 1999) and humans (Eriksson et al, 1998). Adult neurogenesisappears to be functional since abolishing it affects hippocampal-dependent learning (VanPraag et al, 2002; Sahay and Hen 2007). In rodents, stress decreases neurogenesis whileantidepressants (Dranovsky and Hen 2006), environmental enrichment, exercise(Kempermann et al, 1998; van Praag et al, 1999) and learning (Leuner et al, 2004) stimulateneurogenesis. Furthermore, some behavioral effects of pharmacologic antidepressants areblocked by abolishing dentate gyrus (DG) neurogenesis in some species (Santarelli et al,2003; Wang et al, 2008; Surget et al, 2008; David et al, 2009). Neurogenesis decreases withage in different mammals (Rao et al, 2006; Siwak-Tapp et al, 2007; Leuner et al, 2007). Theeffect of fluoxetine on neurogenesis is reported to be age-dependent in Balb/c and C57Bl/6(Navailles et al, 2008) but not 129sv mice (Santarelli et al, 2003).

Dividing cells become neurons in the granule cell layer (GCL) of the DG in the adult humanhippocampus where BrdU immunoreactive (-IR) cells double-label with antibodies specificto neurons (neuron-specific enolase, calbindin, and neuron-specific nuclear protein (NeuN,Eriksson et al, 1998). The human DG consists of the GCL, external molecular layer (ML),and an inner polymorphic layer, the subgranular zone (SGZ, Figure 1a and b). BrdU-IR cellsare localized to the SGZ and inner part of the GCL in rodents (Holick et al, 2007; Santarelliet al, 2003). In adult primates, labeled cells extend to the ML and hilus (Kornack and Rakic1999; Gould et al, 1999). The location of BrdU-IR cells in the human brain is the same as innonhuman primates (Eriksson et al, 1998). A cell cycle and mitosis-related protein, Ki-67(Jakob et al, 2008), labels proliferating cells, and co-localizes with BrdU in the nonhumanprimate (Gould et al, 1999) and rodent (Kee et al, 2002; Reif et al, 2006; Saravia et al, 2007)brain. In rats, BrdU co-localizes with Ki-67 over a 4-day window, making the co-localization timeframe specific (Dayer et al, 2003). In the SGZ of nonhuman primates,BrdU-IR and Ki-67-IR cell densities are positively correlated (Perera et al, 2007). Nestin is aneuron-specific intermediate filament, expressed by quiescent and amplifying neuralprogenitor cells (NPCs, Encinas et al, 2006; Crespel et al, 2005; Takei et al, 2007). Inrodents, quiescent NPCs have a triangular soma in the SGZ and a single or double apicalprocess that extends radially across the GCL, terminating in elaborate arbors of fine leaf-likeprocesses in the ML. Quiescent NPCs undergo asymmetric divisions and generate theamplifying NPCs that propagate in the SGZ through a series of symmetric divisions and exitthe cell cycle within one to three days becoming post-mitotic neuroblasts type 1. Unlikequiescent NPCs, amplifying NPCs do not stain for GFAP or vimentin in mice (Encinas et al,2006). Nestin has been successfully used in human studies to detect NPCs in the brains ofadults and children with temporal lobe epilepsy (Crespel et al, 2005; Takei et al, 2007).

Impaired adult neurogenesis has been hypothesized to be part of the pathogenesis of majordepressive disorder (MDD, Duman et al, 2000; Kempermann and Kronenberg 2003).Neurogenesis is impaired in stress-induced models of depression in rodents (Kempermann

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and Kronenberg 2003; Coyle and Duman 2003; Pham et al, 2003). However, blocking cellreplication by irradiation does not induce depression-like behavior in mice (Santarelli et al,2003). Similarly, inescapable shock decreases cell proliferation, but does not producehelpless behavior in rats (Vollmayr et al, 2003). Therefore, other factors, besides impairedneurogenesis, mediate the development of a depressive phenotype in rodents. Smallerhippocampal volume has been reported in MDD (Sheline et al, 1999; Bremner et al, 2000),together with apoptosis in neuronal cells of the entorhinal cortex, subiculum, DG, CA1, andCA4 regions of the hippocampus (Lucassen et al, 2001). However, in MDD neuron densityin the hippocampus was not reduced (Stockmeier et al, 2004). Therefore it is not clear whythe hippocampus might be smaller in MDD.

We determined the anatomical location within postmortem adult human DG of NPCs(Nestin-IR) and dividing cells (Ki-67-IR). We sought to test the hypotheses thatantidepressant treatment increases NPC number and cell division in the human DG and thatdividing cells are fewer in subjects with MDD. To assess this, we compared the number ofNestin-IR and Ki-67-IR cells in the DG from non-psychiatric controls, untreated MDD, andantidepressant-treated MDD (MDDT) who had received SSRIs or TCAs in the last threemonths of life.

Materials and MethodsBrain Samples

Tissue was obtained from the Brain Collection of the Human Neurobiology Core of theConte Center for the Neuroscience of Mental Disorders and the Macedonia/NYS PsychiatricInstitute Brain Collection. All research was conducted with IRB approval. Brain collectionfollowed a standardized protocol at the time of autopsy. The right hemisphere was cut into 2cm-thick coronal blocks, which were flash-frozen in liquid Freon (-20°C) and stored at-80°C. Samples of tissue from multiple areas of the left hemisphere were fixed in formalinfor neuropathological examination. Brain pH determination (Harrison et al, 1995) andtoxicological analyses were performed on cerebellar samples. Over 30 drugs were screenedfor and quantified if present, including: amphetamine, methamphetamine, methylphenidate,fluoxetine, fluvoxamine, methadone, cocaine, amitriptyline, nortriptyline, imipramine,trimipramine, maprotiline, sertraline, citalopram, chlorimipramine, diazepam, nordiazepam,6-monacetyl morphine, paroxetine, amoxapine, heroin, olanzapine, clozapine, alprazolam,haloperidol, triazolam, buspirone and others. Samples of blood and urine were screened foralcohol, antidepressants, barbiturates, benzodiazepines, cannabis, CO, cocaine, opioids,amphetamine, phencyclidine, salicylates and methadone. A portion of the tissue was fixed informalin for neuropathological examination.

SubjectsNineteen cases were studied: MDD (N = 5) without treatment for more than three monthsprior to death (negative toxicology); MDDT who were on SSRIs (N = 4) or TCAs (N = 3)during the three months prior to death (positive toxicology); and controls, without Axis Ipsychiatric disorder or history of any psychotropic medication for at least three monthsbefore death (C, N = 7, clear toxicology). Subjects (7 females and 12 males) were 17 to 62years of age, and had a postmortem interval (PMI) of 4-24 hours. Four MDD and fiveMDDT cases died by suicide, one MDD and two MDDT cases died from other sudden deathcauses (Table 1). A psychological autopsy was used to obtain DSM-IV Axis I and IIdiagnoses according to the American Psychiatric Association Diagnostic and StatisticalManual of Mental Disorders, fourth edition (DSM-IV, APA, 1994) Life-time information onsuicidal behavior, aggressive impulsive behavior, medical illness, medications, familyhistory and developmental history was obtained (Kelly and Mann 1996). Controls inclusion

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criteria were death by accident, homicide or sudden natural causes, no psychiatric disorderby DSM-IV criteria (APA, 1994) or history of suicide attempts, and negative toxicology forpsychoactive drugs. The inclusion criteria for MDD were: Axis I diagnosis of MDD, majordepressive episode within four months of death and not in remission at death, no history oftreatment in the three months prior to death and negative toxicology for psychoactive drugs.Inclusion criteria for MDDT were the same as for MDD but subjects had to have a history oftreatment for at least the last six weeks of life and positive toxicology for SSRIs or TCAs.Exclusion criteria for all groups were: alcohol or drug dependence or abuse as determinedby the psychological autopsy, positive toxicology for alcohol, liver changes associated withalcoholism, presence of neuropathology, verdict of undetermined death, mental retardation,chronic illness that may affect CNS function (e.g., epilepsy, renal failure, metastaticmalignancy, AIDS), PMI >24 hours, brain not available due to injury and resuscitation withprolonged (>10min) hypoxia.

Tissue ProcessingThe whole hippocampal formation was dissected from two or three consecutive 2 cm-thickfrozen coronal blocks. To optimize the tissue preparation, Bouins, Carnoy's, Zamboni's,paraformaldehyde and glutaraldehyde/paraformaldehyde fixatives were tested and 4%paraformaldehyde was selected as the most suitable for the antibodies used and forpreservation of the integrity of the tissue. After one week fixation at 4°C, tissue wascryoprotected in increasing concentrations of sucrose (up to 30%), sectioned frozen at 50μmon a sliding microtome (Microm HM 440E, Walldorf, Germany), collected into 40-wellboxes, each well containing a series of sections at 2 mm intervals along the anterior-posterior extent of the hippocampal formation, and stored at -20°C in cryoprotectantsolution (30% ethylene glycol in 0.1-M PBS) until use.

ImmunocytochemistryWe used immunocytochemistry to identify NPCs (anti-nestin mouse monoclonal antibody,1:8,000, Chemicon, Temecula, CA); dividing cells (anti-Ki-67 mouse monoclonal antibody,Clone-MM1, 1:200, Novocastra Laboratories Ltd, Newcastle upon Tyne, UK), matureneurons (anti-NeuN mouse monoclonal antibody, 1:100,000; Chemicon) and matureastrocytes (anti-GFAP mouse monoclonal antibody, 1:9,000; Sigma-Aldrich, St. Louis,MO). Primary antibody was omitted in negative control sections. For single labeling,sections were removed from the cryoprotectant and exhaustively washed in 0.01M PBS for60 min, then treated with 0.5% sodium borohydride to remove aldehydes, rinsed in PBS andincubated for 10 min in PBS with 3% hydrogen peroxide to inhibit endogenous peroxidaseactivity. Sets of immediately adjacent sections were processed for Ki-67 or nestin. Primaryantibodies were added to blocking buffer and sections incubated over five days at 4°C. Thisincubation time was used to ensure full penetration of the antibody through the entire 50 μmthickness of the sections, thus allowing 3D stereological cell counting and a correct estimateof the total cell counts. As a secondary antibody, biotinylated horse anti-mouse IgG (1:200,Vectastain Elite ABC, Vector Laboratories Inc., Burlingame, CA) was used. Sections wereprocessed with ABC reagents (Vector Laboratories) and immunostaining visualized with 3′,3-diaminobenzidine (DAB, Sigma-Aldrich). Sections immunoreacted with nestin werestained for Nissl and those assayed with Ki-67 were stained for eosin, in order to highlightneuronal elements. Sections were then dehydrated, cleared and cover-slipped. Doublelabeling experiments were performed to ascertain whether nestin-IR co-localized withNeuN-IR in neurons or GFAP-IR in astrocytes or QNPs, which also stain for GFAP(Encinas et al, 2006). The first antigens were visualized by the ABC reagents and 1% nickelsulfate (Sigma-Aldrich) with 0.05% DAB and 0.01% H2O2 in PBS to yield a blackprecipitate. Then avidin and biotin were blocked using an Avidin/Biotin blocking kit (VectorSP-2001, Vector Laboratories, Jin et al, 2004) and immunocytochemistry for the second

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marker was performed with the same method described, but with 0.05% DAB to yield abrown precipitate. Thus, different antigens were distinguished by their color and appearance,and double-labeled cells were theoretically identifiable.

StereologyWe quantified the number of dividing cells (Ki-67-IR) and NPCs (Nestin-IR) in the DG,which included the SGZ, GCL and ML. The boundaries of the DG were initially defined atlow magnification with a 1.6× objective. Sampling was performed every two mmthroughout the anterior-posterior extent of the DG with a 40× objective for Nestin-IR cellsand a 63× objective for Ki-67-IR cells. Reliability of counting cells with a 40× objective wasobtained counting the Nestin-IR cells with a 40× and a 63× objective in two subjects. Wecounted 6-10 sections per case. Each immunoreacted section was matched using a LeicaWild M3Z type stereoscope (Leica Heerburg, Heerburg, Switzerland) to a near adjacentNissl-stained section that was numbered during the sectioning protocol and served as areference for cytoarchitectonic details. This matching allowed for accurately aligning nestin-and Ki-67-IR sections along the anterior-posterior axis of the DG. We compared the totalnumber of nestin-IR and Ki-67-IR cells across the anterior-posterior axis of the DG in thefour groups (controls, MDD, MDDT who received SSRIs, MDDT who received TCAs). Toestimate the total number of nestin-IR and Ki-67-IR cells we used the optical disectorapproach with the fractionator method (West and Gundersen 1990; Gundersen et al, 1988;Joelving et al, 2006). The counting protocol was done with oversampling, as required whenestimating the number of rare elements (Ngwenya et al, 2005). The upper and lower 3 μm inthe z planes were not counted. The total number of objects counted in the individualdisectors was multiplied by the reciprocals of: sampling fraction for section (SSF), area(ASF) and thickness (TSF). The sampling parameters used to estimate the total cell numberwere: SSF = 0.019, ASF = 0.652, TSF = 0.816. The equipment consisted of a Leica Diaplanmicroscope (Leitz Wetzlar, Germany) equipped with a motorized stage (Ludl ElectronicProducts, Hawthorne, NY) and a CCD color camera (MicroFire CCD, Optronics, Goleta,CA) connected to a computer to run the stereology software (StereoInvestigator, MBFBiosciences Inc., Williston, VT). We measured the volume of the region of interest using theCavalieri's principle, which allows obtaining an estimated volume of an object of arbitraryshape and size. The area of the region of interest within a section was determined by pointcounting and then the volume was calculated by multiplying the sum of the areas of theregion of interest by the mean section thickness, times the distance between each section.We used the following formula:

where V is the volume, T the distance between parallel sections, A the calculated area of asection, and n the total number of sections. The first slide to be sampled was the one inwhich the DG first appeared and subsequent sections were measured every two mmthereafter. All sampling was done by personnel masked to the group assignment of the case.

Statistical AnalysesData analysis was performed using SPSS (16.0 for Mac). An alpha value of 0.05 was usedfor significance level. Different cell types, Ki-67-IR and nestin-IR, were analyzedseparately. Regression analysis on log-transformed data was used to test the effect of age,PMI and pH on cell number within groups. We tested cell number and DG volumedifferences between MDD, MDDT on TCAs, MDDT on SSRIs and controls using ANOVA,with age as covariate when analyzing nestin-IR cells, and Tukey post hoc analysis for pair-

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wise comparisons. We did an age median split and used age ≤ 38 years and age > 38 yearsas factor in a univariate ANOVA with three between-subjects factors: age, sex and group totest the effect of sex on progenitor and dividing cells. Data are expressed as mean ± SEMand p values are 2-tailed.

ResultsAnatomical distribution of progenitor and dividing cells

Nestin-IR cells—Nestin-IR cells were detected in the subgranular zone of the DG, wherethey can be found in niches where multiple cells appear in groups and often showconnections with the vasculature (Figure 1c and 1d). They also appear isolated along theSGZ of the DG. Nestin-IR cells can also be found in the fimbria and in the subsubependymal layer, but those areas were not analyzed in this study. There were no nestin-IR cells in the ML (Figure 1e), CA regions or neocortex. The nestin antibody stains the cell'scytoplasm and processes (Figure 1d). Nestin-IR cells found in the SGZ were multipolar(Figure 1c, 1d), although, less frequently, had a unipolar appearance as they migrated intothe GCL (Figure 1c, arrow). Nestin-IR cells were present throughout the SGZ, in a non-uniform pattern. They appeared distributed along the SGZ, more often in the crest of the DG(Figure 1e), sometimes in groups. Blood vessels also stained for nestin (Figures 1c, 1d, and1e), and NPCs were found adjacent to capillaries, but only in the SGZ of the DG and not inother regions of the hippocampus. In MDDT, Nestin-IR cells exhibited prominent processesand a more complex structure than in untreated MDDs (Figure 2).

GFAP-IR cells—GFAP-IR cells were ubiquitously located throughout the hilus (Figure 3a,3b) and the hippocampal formation. Astrocytes do not stain for nestin, but they do stain forGFAP (Figure 3c, 3d). The nestin/GFAP double labeling show GFAP labels astrocytes thatare distributed in all subregions of the hippocampus (Figure 3a), including the hilus (Figure3b), CA regions, and the adjacent neocortex. GFAP-IR cells located in the SGZ hadprocesses that crossed the GCL and extended into the ML of the DG (Figure 3c). Twodifferent populations of cells in human hippocampus expressed GFAP (Figure 3c, 3d) ornestin (Figure 3e, 3f). A few cells were labeled by both GFAP and nestin.

NeuN-IR neurons—NeuN-IR neurons were observed in the GCL, the hilus and the CAregions (Figure 1a). Dendrites were clearly visible at high magnification (Figure 1b). Doublelabeling experiments (Figure 1e, 1f) showed that nestin-IR cells never stained for NeuN(Figure 1f).

Ki-67-IR cells—Ki-67-IR cells were present in the GCL and the SGZ (Figure 1g). Ki-67 isa nuclear stain; Ki-67-IR cells were identified by a black nucleus (nickel-intensified reactionproduct) and a pink (eosinophilic) cytoplasm (Figure 1h). Occasionally, mitotic figures wereobserved.

Effect of age, sex, PMI and pH on progenitor and dividing cell number and DG volumeIncreasing age was associated with fewer NPCs in the MDDT group (R2 = 0.749, F =11.966, df = 1,4, p = 0.026, Figure 4a), with MDDs and controls showing a similar, notstatistically significant trend (Figure 4b and c; MDD: R2 = 0.596, F = 3.578; df = 1,4; p =0.126; C: R2 = 0.334, F = 2.007; df = 1,4; p = 0.230). Age, used as covariate in a modelcomparing nestin-IR cell number between groups, was associated with fewer total NPCs (F= 5.628; df = 1,17; p = 0.034). We used an age median split (age ≤ 38 years and age >38years) as factor in an ANOVA, including three between-subjects factors: age, sex and group.In the whole sample, age showed a significant effect, subjects ≤ 38 years old had moreNPCs (13 386 ± 155; F = 1.154; df = 1,17; p ≤ 0.001) compared with subjects > 38 years old

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(457 ± 188). Females had more NPCs (11 183 ± 199) than males (6 682 ± 150; F = 13.034;df = 1,17; p = 0.023). To estimate the effect size of age, sex, PMI, pH, brain weight andgroup on NPCs and dividing cells number, we performed univariate ANOVA withcovariates comparing untreated MDD with controls and with MDD treated with SSRIs orTCAs. The results are shown in Table 2.

Dividing cells did not decrease with age in MDDT (R2 = 0.252, F = .271, df = 1,4, p =0.630), MDD (R2 = 0.310, F = 1.348; df = 1,4; p = 0.330) or controls (R2 = 0.010, F =0.040; df = 1,4; p = 0.852, Figure 4d) and were not different in males and females. Neitherage nor sex was related to DG volume. There was no correlation between pH or PMI andNPCs, dividing cells number or DG volume in any group.

Quantification of neural progenitors and dividing cellsNestin-IR cells—Controlling for age and sex, there were more progenitor cells in thetreated MDD group (C: 360 ± 246; MDD: 1 119 ± 752; MDDT: 17 229 ± 3 443; F = 6.935;df = 3,17; p = 0.005) compared with controls and untreated MDD subjects. MDD treatedwith SSRIs had more progenitor cells (Figure 5a, C: 360 ± 246; MDD: 1 119 ± 221;MDDT*TCAs = 14 613 ± 271; MDDT*SSRIs = 19 844 ± 272; F = 984.105; df = 3,17; p ≤0.001) than untreated MDD (p ≤ 0.001) and controls (p ≤ 0.001). NPCs were not different inSSRIs- and TCAs-treated MDDTs (p = 0.169). Without controlling for age or sex, therewere more Nestin-IR cells (C: 360 ± 246; MDD: 1 119 ± 752; MDDT: 17 229 ± 3 443; F =6.935; df = 3,17; p = 0.015) in treated MDDs than in untreated MDDs (p = 0.028) orcontrols (p = 0.038). Dividing the treated MDD group into subjects treated with SSRIs andsubjects treated with TCAs also indicated a significant difference between groups (C: 360 ±246; MDD: 1 119 ± 221; MDDT*TCAs = 14 613 ± 271; MDDT*SSRIs = 19 844 ± 272; F =984.105; df = 3,17; p = 0.038).

Ki-67-IR cells—The number of dividing cells in the treated MDD group was about threetimes greater than in controls, and five times greater than in MDD (C = 20 808 ± 8 301;MDD = 10 944 ± 7 593; MDDT = 57 913 ± 17 045; F = 3.712; df = 2,17; p = 0.049). Thenumber of dividing cells in MDDT receiving TCAs (C = 20 808 ± 8 301; MDD = 10 944 ±7 593; MDDT*TCAs = 94 229 ± 7 494; MDDT*SSRIs = 21 597 ± 8 814; F = 16.243; df =3,17; p ≤ 0.001) was 5.9 times higher than untreated MDD (p ≤ 0.001), 4.3 times higher thanSSRI-treated MDD (p = 0.001) and 4.5 times higher than controls (p ≤ 0.001, Figure 5b).SSRI-treated MDDT subjects had a comparable number of dividing cells to controls (p =0.812). There was half the number of dividing cells in untreated MDD compared withcontrols, but the difference was not statistical significant (p = 0.434).

Nestin-IR and Ki-67-IR cells distribution in the anterior vesus posterior DG—Analysis of the anterior-posterior distribution of NPCs (Figure 5c) and dividing cells (Figure5d) in the DG revealed that the increase in cell number in the treated subjects was moreprominent rostrally. Few cells were found in the most posterior hippocampal sections.

Dentate Gyrus VolumeThe volume (mm3) of the DG was larger in MDDT (C = 106.3 ± 24.4; MDD = 79.6 ± 11.2;MDDT = 188.4 ± 19.1; F = 6.331; df = 2,17; p = 0.009) compared with untreated MDD (p =0.014) and control (p = 0.033). MDDT subjects treated with TCAs (C = 115.4 ± 19.0; MDD= 96.0 ± 19.4; MDDT*TCAs = 220.6 ± 32.4; MDDT*SSRIs = 164.2 ± 34.9; F = 5.450; df =3,17; p = 0.010) had a larger DG volume than untreated MDD (p = 0.011), and controls (p =0.024), but not different from MDDT receiving SSRIs (p = 0.418, Figure 5e). Brain weight(grams) was not different among groups (C = 1 450.0 ± 52.9; MDD = 1 360.0 ± 88.9;MDDT*TCAs = 1 386.6 ± 63.5; MDDT*SSRIs = 1 307.5 ± 66.1; F = .637; df = 3,17; p =

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0.608), as well as section thickness (μm) after processing (C = 32.0 ± 2.4; MDD = 36.7 ±2.9; MDDT*TCAs = 28.8 ± 1.2; MDDT*SSRIs = 36.7 ± 3.1; F = 1.315; df = 3,14; p =0.309). DG volume did not correlate with brain weight (p = 0.732) or section thickness (p =0.218).

DiscussionWe present the first evidence in the human dentate gyrus of more NPCs (Nestin-IR) andmore dividing cells (Ki-67-IR) in SSRI- (sertraline, fluoxetine) or TCA- (nortriptyline,clomipramine) treated MDD compared with untreated MDD or controls. The untreatedMDD group had 50% fewer dividing cells than controls, but the difference was notstatistically significant. The number of NPCs decreased with age. Age was not related to thenumber of dividing cells. Females had more NPCs than males. Overall these results areconsistent with many findings hitherto only observed in rodents.

In rodents, fluoxetine administration for 15 days does not affect division of quiescent NPCsbut increases the number and symmetric divisions of amplifying NPCs (Encinas et al, 2006).The nestin-IR cells counted in the present study did not show vertical processes crossing theGCL and did not end in elaborate arbors in the ML -a characteristic of quiescent (or Type 1)NPCs, at least in rodents- but it is the appearance of the human GFAP-IR cells in this study.The human GFAP-IR cells in this study are either astrocytes (Figure 3d), which areubiquitously located in the hippocampapal formation (Figure 3a) or have the appearance ofquiescent (or Type 1) neural progenitors (Figure 3c), but they do not stain for nestin. Inlower mammals, quiescent NPCs stain both for nestin and GFAP, whereas amplifying (orType 2) NPCs stain for nestin but not for GFAP, and have the morphology of the humannestin-IR cells detected in this study (Encinas et al, 2006). We counted nestin-IR cells thatwere in the SGZ of the human DG and that stained for nestin but not GAFP and had themorphology of amplifying NPCs. These NPCs were often found groups of multiple cellsassociated with the vasculature in the SGZ of the DG, as has been previously described inrats (Palmer et al, 2000, see Figure 2 on page 484). Palmer et al, (2000) suggested this to bea niche where neurogenesis and angiogenesis are linked. Isolated nestin-IR cells were alsofound along the SGZ. In the human, nestin is known to be strongly expressed in newlyformed vascular endothelial cells in the adult heart, pancreas and brain, and its expressiondecreases following cellular differentiation when nestin is replaced by other intermediatefamily proteins (see Salehi et al, 2008). Therefore, nestin immunostaining of blood vesselscannot solely be attributed to poor fixation of the tissue or to poor blood clearance.Additionally, we used hydrogen peroxide to remove endogenous peroxidase activity, and thesame fixed tissue, immunoreacted for NeuN, and Ki-67 did not show any vesselimmunoreactivity.

Given the morphological characteristics of the cells that stain for nestin, antidepressants inthis study appeared to increase amplifying NPCs in human DG, consistent with rodentfindings (Encinas et al, 2006). MDD subjects treated with SSRIs and TCAs had more NPCsthan untreated MDD or controls. TCAs had a more robust effect than SSRIs on dividing cellnumber (Ki-67-IR), and SSRI-treated MDD had a comparable number of dividing cells tocontrols. One previous study used Ki-67 in human tissue to identify dividing cells, and didnot find an effect of antidepressants on Ki-67-IR cell number in anterior humanhippocampal formation (Reif et al, 2006). All the MDD subjects they studied, with theexception of one, were prescribed antidepressant medications. Unfortunately, no toxicologydata were available for those subjects (Torrey et al, 2000), and thus, it is not known whetherthe medications prescribed were actually taken. Fluoxetine, imipramine (Santarelli et al,2003; Malberg et al, 2000), tranylcypromine and reboxetine (Malberg et al, 2000) increaseDG cell proliferation in mice and rats. Inhibition of neurogenesis blocks some behavioral

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effects of antidepressants (Santarelli et al, 2003), suggesting that DG cell proliferation maybe a mechanism of antidepressant action (Santarelli et al, 2003; Duman 2004). Since BDNFexpression increases in the GCL and CA subfields after chronic antidepressantadministration (Nibuya et al, 1995; Dias et al, 2003), action of growth factors may play arole in the observed increased neurogenesis with antidepressant treatment. The functionalrelevance of enhanced neurogenesis in response to antidepressants in man needs to be testedby clinical studies to determine whether the increased cell proliferation is associated withsymptoms improvement in MDD. In our postmortem study a significant proportion of thesubjects died by suicide, raising the possibility that antidepressant treatment in the last sixweeks of life produced cell proliferation but that might not have been sufficient, or notprolonged enough, or even necessary to produce a significant antidepressant effect.Moreover, since neurogenesis is not required for the behavioral effects of environmentalenrichment (Meshi et al, 2006), which has antidepressant-like effects, mechanisms otherthan new cell birth, can produce an antidepressant effect.

The average number of dividing cells in untreated MDD was about half found in controls,but the difference did not reach statistical significance, perhaps because either there is noreal difference or the modest sample size meant insufficient statistical power. Impaired adulthippocampal neurogenesis has been hypothesized to be part of the biological and cellularbasis of major depression (Duman et al, 2000; Kempermann and Kronenberg 2003; Coyleand Duman 2003). Nevertheless, blocking or inhibiting cell proliferation does not inducelearned helplessness in rats (Vollmayr et al, 2003) and does not affect anxiety anddepression-related behaviors in mice (Santarelli et al, 2003; David et al, 2009), raisingdoubts about the etiological role of neurogenesis in major depression.

The increase in neural progenitors and dividing cells in MDDT was localized to the rostralhippocampus, which, in primates (Thierry et al, 2000), is interconnected with the prefrontalcortex, amygdala and nucleus accumbens. The equivalent structure in rodents (Risold andSwanson 1996; Moser and Moser 1998; Sahay and Hen 2007), is the ventral hippocampuswhich has been shown to be involved in anxiety-related behaviors (Sahay and Hen 2007)and to regulate the neuroendocrine responses to psychological stress (Risold and Swanson1996; Nettles et al, 2000). Changes in neurogenesis appear to be subregion-specific fashiondepending on the conditions. Adult animals performing spatial learning tasks have thehighest levels of neurogenesis in dorsal hippocampus (Snyder et al, 2009). Similarly to ourfindings, chronic administration of an antidepressant (agomelatine) increased cellproliferation and neurogenesis specifically in the ventral DG of the rat (Banasr et al, 2006).A functional dissociation of the anterior and posterior axis of the hippocampus has beendemonstrated by lesions studies in rats (Bannerman et al, 2004), and functional studies inprimates (Colombo et al, 1998; Strange et al, 1999; Strange and Dolan 2001). Moreover,anterograde tracer injections studies showed that there is a topographic organization of theintrinsic connections of the DG, and that it is similar in macaque monkey (Kondo et al,2008) and rat (Amaral and Witter 1989). The anatomical specificity of our findings bearsfurther investigation and may link effects on hippocampal function to neuroendocrineresponses to stress.

The NPCs in the DG decrease with increasing age. In rodents, neurogenesis decreases withage (Siwak-Tapp et al, 2007; Rao et al, 2006) and the effect of fluoxetine on DGproliferation is age-dependent (Navailles et al, 2008; Couillard-Despres et al, 2009). Ageaffects antidepressant responsiveness in depression (Keller et al, 1986). Impairedneurogenesis and unavailability of NPCs in older age could be a factor contributing to thepoor antidepressant response observed in some elderly individuals. Increasing age wasassociated with fewer NPCs (Nestin-IR), but age did not affect dividing cells (Ki-67-IR).One possible explanation is that the pool of NPCs decrease with age, while the number of

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Ki-67-IR cells, which labels all dividing cells, including glial and endothelial cells, remainsunaltered. Estrogens affect neurogenesis and stimulate progenitor cell proliferation (Saraviaet al, 2007; Tanapat et al, 1999) and we found more NPCs in females than males, but alarger sample size is needed to confirm this observation. An effect of sex on neurogenesiswould be of particular relevance to mood disorders, where sex differences are pronounced(Oquendo et al, 2007; Kessler et al, 1993).

Ki-67 appears to be a good marker for cell division in the DG and it co-localizes with BrdUin the nonhuman primate (Gould et al, 1999) and rodent (Kee et al, 2002; Reif et al, 2006;Saravia et al, 2007; Reif et al, 2006). In monkeys, there is a positive correlation between thedensity of BrdU- and Ki-67-IR cells in the SGZ (Perera et al, 2007). We found more Ki-67-IR cells than nestin-IR cells in every group. It is possible that a proportion of the dividingcells that we observed here are astroglia, microglia, or endothelial cells. Because of thedifferences in the timing of the expression of the proteins we cannot identify cells doublelabeled for Ki-67 and NeuN (Kee et al, 2002). Double-labeling with BrdU and NeuN wasdemonstrated when BrdU was injected antemortem in rodents and 75–90% of BrdU-IR cellseventually expressed NeuN (Madsen et al, 2000). Moreover, Ki-67, as BrdU is alsoexpressed during apoptosis (Kuan et al, 2004). Therefore, Ki-67-IR cell number may not beas good a surrogate of neurogenesis as nestin-IR cell number in the human DG. In keepingwith this idea we observed differences between nestin and Ki-67 both in the response toSSRIs and in the age effect. The fact that no effect of age is observed with Ki-67 could beexplained by this marker labeling not only neural precursors, but all dividing cells.

The volume of the DG was larger in MDDT compared with untreated MDD and controlgroups. MDD treated with TCAs or SSRIs showed similar DG volume. We did not finddifferences in brain weight or post-processing section thickness among groups, suggestingthat the observed larger DG volume in MDDT was not attributable to a larger brain or lessshrinkage with fixation. Some imaging studies in vivo found smaller hippocampal volume inMDD compared to healthy controls (Sheline et al, 1999; Bremner et al, 2000; McKinnon etal, 2009) but effects of treatment are less studied. The increased DG volume in MDDT maysuggest that antidepressant treatment increases total mature granule cell number, or volumeof the neuropil. The animal model of stress-induced depression is associated with cell deathand dendritic shrinkage in the hippocampal formation, which is reversed by antidepressanttreatment (McEwen 1999; Moore et al, 2000). Inescapable foot shock caused loss of spinesynapses selectively in CA1, CA3, and DG, reversed by desipramine (McEwen 1999; Mooreet al, 2000). One previous study showed higher density of granule cells and glia in the DGand CA regions and smaller soma of pyramidal neurons in MDD compared with controls,suggesting that a reduction in neuropil may account for smaller hippocampal volume inMDD (Stockmeier et al, 2004). The observed larger volume of the DG and the higher NPCnumber in MDDT could be attributable not only to the increased neurogenesis, but also to anincrease of cell survival resulting from antidepressants increasing Bcl-2 (Peng et al, 2008;Fricker et al, 2005), and BDNF (Nibuya et al, 1995; Dias et al, 2003), which also regulatescell survival (Sairanen et al, 2005; Benraiss et al, 2001). Whether potential antidepressanteffects on neurogenesis or neuropil can change the hippocampus volume and how thataffects the course of MDD remains to be determined.

We did not find an effect of PMI or pH on cell number. PMI could affect brain antigenicity(Lewis 2002; Li et al, 2003), and thus, immunocytochemistry, while the effect of pH onantigenicity is not known. We limited the potentially deleterious effects of PMI and pH byusing samples with a PMI ≤ 24 hours and pH > 6.0.

The primary limitation of this study is the small sample size. Larger samples should be usedfor replication of the observations and to determine whether the changes in neurogenesis are

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present in untreated MDD. Also, comparing subjects who died by suicide with subjects whodid not may help determine the effect of stress on neurogenesis in human. We used everyprotocol-eligible case at the time of the study and are collecting other cases for futurestudies. The possibility of a difference in MDD severity between treated and untreatedMDDs should also be considered. Treated MDDs may have been more severely ill thanuntreated MDDs and therefore more likely to have sought or been prescribed treatment. Onthe other hand, the high percentage of suicide cases in the untreated MDD group raises thepossibility that this group was untreated because of poor compliance or because of failure toaccess treatment. Finally, the fact that antidepressant exposure is associated with evidence ofmore neurogenesis does not mean it is the mechanism of antidepressant action. Futurestudies must determine whether degree of antidepressant response is linked to increasedneurogenesis, and if it is, would motivate seeking new treatments that have this property.

AcknowledgmentsMihran J. Bakalian assisted with graph preparation and data management and Jennifer Lau with stereologicalanalysis. Work was supported by PHS grants MH40210, MH62185, MH64168 and MH083862, the AmericanFoundation for Suicide Prevention, the Diane Goldberg Foundation and the Janssen Fellowship in TranslationalNeuroscience.

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Thierry AM, Gioanni Y, Degenetais E, Glowinski J. Hippocampo-prefrontal cortex pathway:anatomical and electrophysiological characteristics. Hippocampus. 2000; 10:411–419. [PubMed:10985280]

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Van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD, Gage FH. Functional neurogenesis in theadult hippocampus. Nature. 2002; 415:1030–1034. [PubMed: 11875571]

Vollmayr B, Simonis C, Weber S, Gass P, Henn F. Reduced cell proliferation in the dentate gyrus isnot correlated with the development of learned helplessness. Biol Psychiatry. 2003; 54:1035–1040.[PubMed: 14625145]

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Figure 1. Immunocytochemistry for NeuN, nestin, double labeling for NeuN and nestin andimmunocytochemistry for Ki-67 in human dentate gyrus (DG) from a 57 years old femalecontrol (a-f) and a 75 years old male control (g-h) who were not on medication(a) NeuN-immunoreactive (-IR) cells in the DG, hilus and CA regions of the hippocampus.(b) The subgranular zone (SGZ), granule cell layer (GCL) and Molecular layer (ML) areindicated; at higher magnification, the initial segments of the dendrites of the neurons of thehilus are clearly stained for NeuN (the same cells in the black box in a). (c) Nestin-IR cells(brown) along the SGZ of the DG exhibiting their characteristic multipolar appearance. Noteone unipolar cell (arrow), which appears to have a higher level of differentiation, locatedwithin the GCL. Section is stained for Nissl with Cresyl Violet. (d) Differential interference

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contrast image of a nestin-IR cell, showing the immunostained perikarya and multipleprocesses, including one touching a blood vessel. (e) Double labeling of NeuN-IR granulecells (in black) and nestin-IR cells (in brown). Blood vessels are stained for nestin. (f) Thereis no co-labeling of Nestin and NeuN (the same cells in the black box in e). (g) Ki67-IR cells(nucleus in purple) in the GCL and SGZ of the DG. (k) Differential interference contrastimage of the same cells in the black box in g. Four nuclei are labeled. Afterimmunocytochemistry, sections were stained with eosin to label cytoplasm withoutinterfering with the Ki-67 nuclear stain.

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Figure 2. Nestin-immunoreactive (-IR) cells and vessels in the dentate gyrus (DG) from a 29years old male with Major Depressive Disorder (MDD) who was not on medication (a) and a 31years old male MDD who was treated with fluoxetine (b)(a) The subgranular zone (SGZ) and granule cell layer (GCL) are indicated. Cells are stainedfor Nissl with Cresyl Violet. Nestin-IR cells and vessels appear in brown. (b) Thefluoxetine-treated MDD shows more prominent nestin-IR cells processes and vesselscompared to the untreated MDD (in a).

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Figure 3. Human dentate gyrus (DG) treated with double-labeling immunocytochemistry fornestin and glial fibrillary acid protein (GFAP) and stained for Nissl substance. The subject was a57-year-old female control with no medication(a) The granular cell layer (GCL) appears in blue, GFAP-immunoreactive (-IR) cells stainedwith diaminobenzidine (DAB) appear brown. GFAP labels astrocytes in all subregions ofthe hippocampus (b) GFAP-IR cells are ubiquitously located throughout the hilus. (c)GFAP-IR cells located in the subgranular zone (SGZ) show processes that extend toward themolecular layer (ML), crossing the GCL. A GFAP-IR cell is seen in the SGZ near the loweredge of the picture (arrow). (d) GFAP-IR cells in the hilus at higher magnification.

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Astrocytes do not stain for nestin, but they do stain for GFAP (e, f) Nestin-IR cells (in black)appear along the SGZ (arrows).

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Figure 4. Progenitor cell (Nestin-IR) number decreased with increasing age(a) Increasing age was associated with fewer progenitor cells (Nestin-IR) in subjects withMajor Depressive Disorder (MDD) who received selective serotonin reuptake inhibitors(MDDT*SSRI) or tryciclics (MDDT*TCA). (b, c) In controls (C) and in untreated MDDsubjects, the decrease in progenitor cell number with age did not reach statisticalsignificance. (d) Age did not affect the number of dividing cells (Ki-67-IR) in any of thegroups.

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Figure 5. Neural progenitor and dividing cells are increased in the dentate gyrus (DG) ofsubjects with Major Depressive Disorder (MDD) who were treated with antidepressants(MDDT) compared to untreated MDDs and controls (C)(a) Progenitor cells (Nestin-IR) were higher in MDDT treated with tryciclics(MDDT*TCA), as well as in MDDT treated with selective serotonin reuptake inhibitors(MDDT*SSRI) compared to untreated MDD and C (F = 984.105; df = 3,17; p ≤ 0.001). (b)Dividing cells (Ki-67-IR) were higher in MDD*TCA compared to the other groups (F =16.243; df = 3,17; p ≤ 0.001). (c) Number of progenitor cells (Nestin-IR) in sections locatedin the anterior versus posterior hippocampal formation (error bars represent S.D.) (d)Number of dividing cells (Ki-67-IR) in sections located in the anterior versus posterior

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hippocampal formation (error bars represent S.D.) (e) DG volume in MDDT subjects treatedwith TCAs was larger compared to controls and MDD (F = 5.450; df = 3,17; p = 0.010) butnot different from MDDT receiving SSRIs. (f) Image of one of the hippocampus sectionsshowing the DG stained for Nissl (in blue). The outline defines the region of interest, withinwhich cells were counted using stereology.

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Boldrini et al. Page 24

Tabl

e 1

Dem

ogra

phic

and

clin

ical

cha

ract

eris

tics

of

subj

ects

Gro

upA

geSe

xP

MI

(h)

Bra

in p

HC

ause

of

deat

hA

xis

I &

II

diag

nosi

sT

oxic

olog

yM

edic

atio

ns p

resc

ribe

d (L

ast

3m

onth

s)

C17

Fem

ale

226.

57M

VA

-pas

seng

erN

one

Lid

ocai

neN

one

C41

Fem

ale

156.

84H

omic

ide

(gun

sho

t wou

nd)

Non

eC

lear

Non

e

C50

Fem

ale

226.

94Pe

rito

nitis

Non

eC

lear

Non

e

C25

Mal

e9.

56.

87St

abbi

ng (

acci

dent

)N

one

CO

Non

e

C31

Mal

e10

6.24

MV

A-d

rive

rN

one

Lid

ocai

ne D

iaze

pam

Tra

mad

olN

one

C34

Mal

e12

6.38

Acu

te m

yoca

rdia

l inf

arct

Non

eL

idoc

aine

Thy

roxi

ne

C53

Mal

e16

6.24

Hom

icid

e (s

tabb

ing)

Non

eC

lear

Non

e

MD

D34

Fem

ale

46.

93Su

icid

e (h

angi

ng)

MD

DC

lear

Non

e

MD

D29

Mal

e15

6.73

Suic

ide

(han

ging

)M

DD

Cle

arN

one

MD

D29

Mal

e22

6.34

Acu

te b

ronc

hial

ast

hma

MD

DT

heop

hylli

neT

heop

hylli

ne

MD

D56

Mal

e21

6.56

Suic

ide

(han

ging

)M

DD

Cle

arN

one

MD

D62

Mal

e21

6.37

Suic

ide

(dro

wni

ng)

MD

DC

lear

Lor

azep

am

MD

DT

*SSR

I24

Fem

ale

146.

87Su

icid

e (f

all f

rom

hei

ght)

MD

D B

UL

/AN

OR

Sert

ralin

e B

upro

pion

Fluo

xetin

e T

razo

done

Bup

ropi

onC

lona

zepa

m

MD

DT

*SSR

I50

Fem

ale

247.

01A

cute

res

pira

tory

fai

lure

MD

DSe

rtra

line

Dip

henh

ydra

min

eSe

rtra

line

MD

DT

*SSR

I31

Mal

e6

6.61

Suic

ide

(gun

sho

t wou

nd)

MD

DFl

uoxe

tine

Nor

prop

oxyp

hene

Fluo

xetin

e A

mitr

ipty

line

MD

DT

*SSR

I61

Mal

e24

7.14

Suic

ide

(fal

l fro

m h

eigh

t)M

DD

Fluo

xetin

e Se

rtra

line

Bar

bitu

rate

sA

ceto

min

ophe

nFl

uoxe

tine

Sert

ralin

e B

arbi

tura

tes

Salic

ylat

es

MD

DT

*TC

A28

Fem

ale

196.

32Su

icid

e (f

all f

rom

hei

ght)

MD

D B

UL

/AN

OR

Nor

trip

tylin

eN

ortr

ipty

line

Clo

naze

pam

MD

DT

*TC

A38

Mal

e9

6.39

Acc

iden

tal o

verd

ose

MD

D C

lust

er B

PD

Clo

mip

ram

ine

Dia

zepa

m D

oxep

inSe

rtra

line

Flur

azep

am

MD

DT

*TC

A61

Mal

e12

6.76

Suic

ide

(fal

l fro

m h

eigh

t)M

DD

Nor

trip

tylin

e Pe

rphe

nazi

neN

ortr

ipty

line

Ven

lafa

xine

Ser

tral

ine

Alp

razo

lam

Per

phen

azin

e

C: C

ontr

ols;

MD

D: M

ajor

Dep

ress

ive

Dis

orde

r; M

DD

T: T

reat

ed s

ubje

cts

with

Maj

or D

epre

ssiv

e D

isor

der;

SSR

I: S

elec

tive

sero

toni

n re

upta

ke in

hibi

tor;

TC

A: T

ricy

clic

ant

idep

ress

ant;

MV

A: M

otor

veh

icle

acci

dent

; BU

L/A

NO

R: E

atin

g D

isor

der

(Bul

imia

, Ano

rexi

a); P

D: P

erso

nalit

y D

isor

der;

CO

: Car

bon

mon

oxid

e. (

Subj

ects

wer

e as

sign

ed to

the

MD

DT

*SSR

I or

the

MD

DT

*TC

A g

roup

acc

ordi

ng to

the

drug

dete

cted

in th

e br

ain

toxi

colo

gica

l exa

m, s

ince

med

icat

ion

pres

crib

ed is

not

nec

essa

rily

take

n by

the

patie

nt.)

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Boldrini et al. Page 25

Tabl

e 2

Sour

ces

of v

aria

tion

of

neur

al p

roge

nito

r ce

ll (n

esti

n-IR

) an

d di

vidi

ng c

ells

(K

i-67

-IR

) nu

mbe

r in

unt

reat

ed M

DD

com

pare

d to

con

trol

s an

dM

DD

tre

ated

wit

h tr

icyc

lics

(MD

D*T

CA

) or

SSR

Is (

MD

D*S

SRI)

ant

idep

ress

ants

Nes

tin-I

R c

ells

MD

D v

s co

ntro

lsM

DD

vs

MD

D*T

CA

MD

D v

s M

DD

*SSR

I

F1,

10P

η2F

1,16

Pη2

F1,

19P

η2

Fac

tor

Dia

gnos

is27

.268

.014

.901

29.6

66.0

12.9

088.

411

.044

.678

Sex

10.3

16.0

49.7

7515

.007

.030

.833

.243

.656

.075

Cov

aria

tes

Age

1.07

2.3

77.2

631.

066

.378

.262

1.82

0.2

49.3

13

PMI

3.58

7.1

55.5

451.

947

.396

.661

26.9

25.1

21.9

64

pH2.

529

.210

.457

4.20

3.2

89.8

081.

880

.242

.320

Bra

in W

eigh

t7.

966

.106

.799

6.12

1.1

32.7

54.5

85.5

84.3

69

Ki-

67-I

R c

ells

MD

D v

s co

ntro

lsM

DD

vs

MD

D*T

CA

MD

D v

s M

DD

*SSR

I

F1,

10P

η2F

1,10

Pη2

F1,

10P

η2

Fac

tor

Dia

gnos

is64

.476

.077

.985

1489

.35

.001

.999

99.2

01.0

64.9

90

Sex

19.1

33.1

43.9

50.9

73.4

28.3

274.

774

.272

.828

Cov

aria

tes

Age

.044

.869

.042

8.83

8.2

07.8

98.0

62.8

27.0

30

PMI

3.10

5.3

29.7

563.

035

.332

.752

.328

.625

.141

pH4.

568

.279

.820

.917

.514

.478

5.09

2.2

66.8

36

Bra

in W

eigh

t.0

90.7

93.0

43.0

66.0

81.9

923.

349

.318

.770

MD

D, m

ajor

dep

ress

ive

diso

rder

; SSR

I, s

elec

tive

sero

toni

n re

upta

ke in

hibi

tor;

PM

I, p

ost-

mor

tem

inte

rval

. Num

bers

in b

old

repr

esen

t sig

nifi

cant

res

ults

. Num

bers

in it

alic

rep

rese

nt r

esul

ts w

ith a

tren

d fo

rsi

gnif

ican

ce.

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